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| Box: Sequencing methods |
- Maxam-Gilbert Sequencing: This method brings about chemical modification of the bases and also requires that the DNA sequence be labeled radioactively at the 5’end. Chemical modification is brought about in such a way that four different types of modificed products are generated. The final sequence is deduced by running a SDS-Polyacrylamide gel with the four products in four parallel lanes, which is followed by exposing the gel to X-ray film for autoradiography. Technique is limiting with respect to the use of radioactive labels.
- Sanger Sequencing: This is also known as the chain termination method, which relies on the fact that; a modified base (for instance dideoxynucleotides instead of the deoxynucleotides) when incorporated during in vitro replication causes the process to cease at that point. Each of the four modified bases are labeled with four different fluorescent labels, which are further detected in automated sequencers.
- The advent of numerous advancements and improvements in the genomics field has led to the development of high-throughput sequencing methods, which have made it possible that thousands of genes be sequenced at a time. These techniques mainly include: Pyrosequencing, Shot gun Sequencing, Sequencing by synthesis, etc.
- Currently used sequencing techniques also employ Bacterial Artificial Chromosomes (BACs) which are DNA constructs that are useful for cloning purposes. These cloning vectors can carry DNA inserts of around 150-350 kbp and have been extremely useful in various genome- sequencing projects carried out. The genomic DNA is cleaved using suitable restriction endonuclease and inserted into the bacterial artificial chromosome. The amplified sequences are sequenced using an automated sequencer and then mapped by aligning the overlapping fragments to obtain the original DNA sequence. The information obtained through these sequencing projects is documented in genome databases and are extremely useful in correlating gene and protein sequences.
- The use of Next-Gen sequencing platforms has facilitated genomic studies to be carried out on a large scale and has further expanded the scope of generating information about the structural and functional aspects of various genes. The plethora of attributes associated with the different coding genes can be explored only when the genomic data is supplemented with data obtained from proteomic studies.
- Third Generation Sequencing Technology relies on Scanning Tunnel Electron Microscopy, Fluorescence Resonance Energy Transfer, Single Molecule Real Time Sequencing and Protein nanopores.
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